EP3630805A1 - Mic-1 compounds and uses thereof - Google Patents

Mic-1 compounds and uses thereof

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Publication number
EP3630805A1
EP3630805A1 EP18730247.6A EP18730247A EP3630805A1 EP 3630805 A1 EP3630805 A1 EP 3630805A1 EP 18730247 A EP18730247 A EP 18730247A EP 3630805 A1 EP3630805 A1 EP 3630805A1
Authority
EP
European Patent Office
Prior art keywords
mic
seq
chem
amino acid
formula
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP18730247.6A
Other languages
German (de)
English (en)
French (fr)
Inventor
Xiang Gao
Xujia Zhang
Hongtao GUAN
Henning Thogersen
Kristian Sass-Orum
Lars Fogh Iversen
Per Norgaard
Sebastian Beck JORGENSEN
Kristian Tage Hansen
Yi Wang
Kilian Waldemar Conde FRIEBOES
Birgit Wieczorek
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Novo Nordisk AS
Original Assignee
Novo Nordisk AS
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Publication date
Application filed by Novo Nordisk AS filed Critical Novo Nordisk AS
Publication of EP3630805A1 publication Critical patent/EP3630805A1/en
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to MIC-1 compounds and their pharmaceutical use. INCORPORATION-BY-REFERENCE OF THE SEQUENCE LISTING
  • MIC-1 Macrophage Inhibitory Cytokine-1 (MIC-1) was first described in 1997 (Bootcov et al, Proc. Natl. Acad. Sci. Oct 1997) based on experiments showing increased expression in activated macrophages. MIC-1 has subsequently been identified by others and given several additional names such as placental transforming growth factor beta (PTGF- ⁇ ), placental bone morphogenetic protein, growth differentiation factor-15
  • MIC-1 is a distant member of the TGF-beta super family, a family of peptide hormones involved in cell growth and differentiation. MIC-1 circulates as a cysteine-rich homodimer with a molecular mass of 24.5 kDa. Human wild-type MIC- 1 has a short half-life, meaning that treatment with wt-MIC-1 requires daily
  • WO 2005099746 concerns a method of modulating appetite and/or body weight by administering a MIC-1 modulating agent.
  • the present invention relates to MIC-1 compounds comprising a MIC-1 polypeptide with an N-terminal amino acid extension and a protractor attached to the amino acid extension.
  • the MIC-1 compounds of the invention comprise a MIC-1 polypeptide with an N-terminal amino acid extension and a protractor attached to the amino acid extension, wherein the amino acid extension comprises 3 to 200 amino acid residues, and the MIC-1 polypeptide with the amino acid extension has a calculated pi lower than 6.5.
  • the MIC-1 compounds of the invention have an N- terminal amino acid extension with one Cysteine residue, wherein the protractor is attached to the Cysteine residue.
  • the protractor comprises, or consists of at least one of each of Chem. 1, Chem. 2, Chem. 3 and Chem. 4; wherein Chem. 1 is selected from the group consisting of:
  • Chem. 1C HOOC-benzene-0-(CH 2 ) y -CO- Chem. ID (lH-tetrazol-5-yl)-(CH 2 ) x -CO- wherein x integer in the range of 12-20,
  • Chem. 2 is selected from the group consisting of:
  • Chem. 2A *-(NH-CH(COOH)-(CH 2 )m-CO) k *,
  • n of Chem. 2 is an integer in the range of 1-5, and
  • k of Chem. 2 is an integer in the range of 0-4; wherein Chem. 3 is
  • k of Chem. 3 is an integer in the range of 1-10,
  • n is an integer in the range of 1-5, and
  • Chem. 4A *-NH-(CH 2 ) m -NH-CO-CH 2 -*,
  • Chem. 4B *-NH-CH(COOH)-(CH 2 ) m -NH-CO-CH 2 -*,
  • Chem. 4D o wherein m of Chem. 4 is an integer in the range of 1-5, and n is an integer in the range 2-6; and wherein Chem. 1, Chem. 2, Chem. 3, and Chem. 4 are interconnected via amide bonds, connecting the*-NH end of a Chem. to the CO-* end of another Chem., and wherein Chem. 4 is connected to the sulphur atom of the Cysteine residue of the amino acid extension.
  • the MIC-1 compounds of the invention comprise an N- terminal extension that has surplus of acidic amino acid residues (Aspartic acid and/or Glutamic acid) of at least 3, 4, 5 or 6 compared to the number of basic amino acid residues (Lysine, Arginine and/or Histidine).
  • the MIC-1 compounds comprise N- terminal extensions composed of amino acid residues selected among the group consisting of A, C, E, G, P, S, T, Q, N and D, wherein the amino acid extension comprises at least three E and/or D amino acid residues.
  • the MIC-1 compounds of the invention comprise an MIC-1 polypeptide that display at least 85%, 90%, 95% or 98% sequence identity to MIC-1 of SEQ ID NO : l.
  • the MIC-1 compounds of the invention comprise an MIC-1 polypeptide that comprises a deletion of the first three residues (MIC-l-Al-3) or a deletion of Asparagine 3 (des-N3) compared to MIC-1 of SEQ ID NO : l .
  • the MIC-1 compound comprises a MIC-1 polypeptide and an N-terminal amino acid extension with an amino acid sequence according to SEQ ID NO : 87, 90, 92, 93, 94, 97, 98, 99, 100, 101, 102, 108, 109, 111, 112, 113, 114, 115, 116, 117, 164, 288, 289, 290, 291 or 292.
  • the MIC-1 compounds of the invention have retained MIC-1 receptor potency and in vivo efficacy on lowering food intake and body weight. These MIC-1 compounds can therefore be used for treatment of metabolic disorders such as obesity, diabetes, cardiovascular diseases like dyslipidaemia and arteriosclerosis and other disorders such as steatohepatitis and diabetic nephropathy.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the MIC-1 compound of the invention or a pharmaceutically acceptable salt, amide or ester thereof, and one or more pharmaceutically acceptable excipients.
  • the invention provides a MIC-1 compound for use in the prevention and/or treatment of a metabolic disorder, wherein the metabolic disorder is obesity, type 2 diabetes, dyslipidemia, or diabetic nephropathy. In one aspect, the invention provides a MIC-1 compound for use in the prevention and/or treatment of dyslipidaemia, arteriosclerosis, non-alcoholic nephropathy.
  • the MIC-1 compounds of the invention have a protracted plasma exposure, i.e. a prolonged half-life compared to human wild type MIC-1.
  • the MIC-1 compounds of the invention have improved solubility. In one aspect, the MIC-1 compounds of the invention have improved chemical stability.
  • Fig.l The expression of MIC-1 polypeptides with N-extensions with single 12- mer building blocks. All cells were grown in TB at 37 °C and proteins were induced to express by adding 0.5mM IPTG after OD600 reached 1.0. Cells were harvested after overnight and the expression level was checked by loading the total lysate on SDS-PAGE. Wild type human MIC-1 (MIC-1) was loaded as the positive control.
  • Fig.2 The expression of MIC-1 polypeptides with N-extensions with double 12- mer building blocks. All cells were grown in TB at 37 °C and proteins were induced to express by adding 0.5mM IPTG after OD600 reached 1.0. Cells were harvested after overnight and the expression level was checked by loading the total lysate on SDS-PAGE. MIC-1 was loaded as the positive control.
  • Fig.3 a) Comparison of expression levels among MIC-1 polypeptides with N- extensions initiating with 12mer- (4+2+_), - (4+4+_) and - (4+3+_). It should be noticed that the group bearing 12mer-(4+3_) and the construct indicated by the dot contain M57L in the MIC-1 polypeptide sequence, b) the effects of the extended 12mers on the expression level. In addition, the lowest data point in the group of 3.6 is the MIC- 1 polypeptide containing M57L. In this figure, "1.6 latter" represents TSTEEG, "2.6” represents TSESAT, "3.6” represents TSTEPS and "4.6” represents SEPATS.
  • Fig.4 SDS-PAGE of representatives bearing 12mer-(4+2+_), 12mer- (4+3+_) + M57L, 12mer-(three repeats) and 12mer-(four repeats).
  • T total protein
  • S soluble fraction
  • P cell pellet (inclusion body).
  • Fig.5 MIC-1 polypeptides with in-sequence mutations.
  • the MIC-1 polypeptide sequence is MIC-1 ⁇ 1-3.
  • T total protein
  • S soluble fraction
  • P cell pellet (inclusion body).
  • Fig.6 Body weight change over time during Pharmacodynamic (PD) study in pigs. 01 : Compound 01 ; 05: Compound 05 DETAILED DESCRIPTION
  • the invention relates to MIC-1 compounds comprising a MIC-1 polypeptide with an N-terminal amino acid extension and a protractor attached to the amino acid extension.
  • the MIC-1 compounds of the invention comprise a MIC-1 polypeptide an N-terminal amino acid extension and a protractor attached to the amino acid extension, wherein the amino acid extension comprises 3 to 200 amino acid residues, and the MIC-1 polypeptide and the amino acid extension together have a calculated pi lower than 6.5.
  • the MIC-1 compounds of the invention are biologically active. For example, they are potent, retain full efficacy compared to MIC-1 and also, they have a protracted plasma exposure profile, i.e. have a pronged half-life. The particular combination of potency and long half-life is desirable.
  • MIC-1 Macrophage Inhibitory Cytokine-1
  • GDF-15 Growth Differentiation Factor 15
  • MIC-1 is synthesized as a 62 kDa intracellular homodimer precursor protein which subsequently is cleaved by a furin-like protease into a 24.5 kDa homodimer.
  • the sequence of the full length wild type human MIC-1 is available from the UNIPROT database with accession no. Q99988.
  • the 308 amino acid precursor sequence includes a signal peptide (amino acids 1-29), a propeptide (amino acids 30-196) and a MIC-1 monomer sequence (amino acids 197-308).
  • the 112 amino acid MIC-1 monomer sequence is included herein as SEQ ID NO : l .
  • MIC-1 monomer contains nine cysteine residues which give rise to the formation of 4 intrachain disulphide bonds and one interchain disulphide bond to create a covalently linked 24.5 kDa homodimer.
  • a naturally occurring mutation corresponding to H6D in the MIC-1 monomer sequence (SEQ ID NO : l) has been described.
  • MIC-1 compound refers to a compound comprising a MIC-1 polypeptide, an N-terminal amino acid extension, and a protractor.
  • the MIC-1 compound is typically in the form of a homodimer.
  • MIC-1 polypeptide refers to the human MIC-1 monomer sequence of SEQ ID NO : l or an analogue thereof. Numerical references to particular MIC-1 residues, if not stated otherwise, refer to the 112 amino acid monomer sequence (i.e., residue 1 is Alanine (Al), and residue 112 is Isoleucine (1112).
  • MIC-1 analogue refers to a MIC-1 polypeptide, which is an amino acid variant of the monomer MIC-1 sequence of SEQ ID NO: l .
  • a MIC-1 analogue is a MIC-1 polypeptide in which a number of amino acid residues have been changed when compared to human MIC-1 (SEQ ID NO: 1). These changes may represent, independently, one or more amino acid substitutions, additions, and/or deletions.
  • MIC-1 analogues may be described by reference to the amino acid residue which is changed, the number of the amino acid residue (i.e. the corresponding position in the MIC-1 monomer sequence (SEQ ID NO : l)), and the change (e.g. the amino acid residue change to).
  • the MIC-1 analogue is a functional variant of the MIC-1 of SEQ ID NO : l.
  • the MIC-1 analogues display at least 85%, 90% or 95% sequence identity to MIC-1 of SEQ ID NO : l .
  • the two peptides H6D MIC-1 and MIC-1 of SEQ ID NO: 1 are aligned.
  • the sequence identity of the H6D MIC-1 analogue relative to MIC-1 of SEQ ID NO : 1 is given by the number of aligned identical residues divided by the total number of aligned residues in MIC-1 of SEQ ID NO: 1.
  • sequence identity in percentage is (112-1)/112 X 100.
  • the N-terminal amino acid extension is not included.
  • a suitable alignment program can be tested with a suitable alignment program "needle", which is a Needleman-Wunsch alignment. The algorithm for this alignment program is described in Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48: 443-453.
  • the MIC-1 analogues comprise less than 15, 10 or 5, amino acid modifications (substitutions, deletions, additions (including insertions) and any combination thereof) relative to human MIC-1 of SEQ ID NO: l .
  • amino acid modification used throughout this application is used in the meaning of a modification to an amino acid as compared to monomer MIC-1 (SEQ ID NO : l). This modification can be the result of a deletion of an amino acid, addition of an amino acid, substitution of one amino acid with another or a substituent covalently attached to an amino acid of the peptide.
  • amino acids may be substituted by conservative substitution.
  • conservative substitution denotes that one or more amino acids are replaced by another, biologically similar residue. Examples include substitution of amino acid residues with similar characteristics, e.g. small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids and aromatic amino acids.
  • amino acids may be substituted by non-conservative substitution.
  • non-conservative substitution denotes that one or more amino acids are replaced by another amino acid having different characteristics. Examples include substitution of a basic amino acid residue with an acidic amino acid residue, substitution of a polar amino acid residue with an aromatic amino acid residue, etc.
  • the non-conservative substitution is substitution of a coded amino acid to another coded amino acid having different characteristics.
  • the MIC- 1 analogues may comprise substitutions of one or more unnatural and/or non-amino acids, e.g ., amino acid mimetics, into the sequence of MIC- 1.
  • the asparagine in the position corresponding to position 3 of monomer MIC- 1 sequence is substituted to Serine (N3S), Glutamic acid (N3E), Alanine (N3A), or Glutamine (N3Q) .
  • the asparagine in the position corresponding to position 3 of human MIC- 1 monomer sequence is substituted to Glutamic acid (N3E) .
  • the arginine in the position corresponding to position 2 of human MIC-1 monomer sequence (SEQ ID NO : l) has been substituted to alanine (R2A), and the asparagine in the position corresponding to position 3 of human MIC- 1 monomer sequence (SEQ ID NO : l) has been substituted to Glutamic acid (N3E) .
  • the arginine in the position corresponding to position 2 of human MIC-1 monomer sequence (SEQ ID NO : l) has been substituted to Glutamic acid (R2E), and the asparagine in the position corresponding to position 3 of human MIC- 1 monomer sequence (SEQ ID NO : l) has been substituted to Serine (N3S) .
  • the MIC- 1 analogues of the invention may have one or more amino acid residues deleted from the amino acid sequence of MIC- 1 (SEQ ID NO : l), alone or in combination with one or more insertions or
  • MIC- 1 analogues with amino acid deletions may be described by "des", reference to the amino acid residue which is deleted, and followed by the number of the deleted amino acid (i.e. the corresponding position in the monomer MIC- 1 (SEQ ID NO : l)) .
  • the asparagine in the position corresponding to position 3 of human monomer MIC- 1 (SEQ ID NO : l) is deleted (MIC- 1 des-N3, SEQ ID NO : 2) .
  • the alanine in the position corresponding to position 1 of human monomer MIC- 1 (SEQ ID NO : l) is deleted (MIC- 1, des-Al) .
  • MIC- 1 analogues with a truncation of one or more amino acid residues at the N or C terminal may be described by "MIC- 1- ⁇ " and reference to the number(s) of the deleted amino acid residues (i.e. the corresponding position in the monomer MIC- 1 (SEQ ID NO : 1)) .
  • the first three residues (Al, R2, N3) at the N terminal are deleted ( ⁇ - ⁇ 1-3, SEQ ID NO : 3) .
  • Insertions In one aspect, the MIC-1 analogues of the invention have one or more amino acid residues inserted into the amino acid sequence of human MIC-1, alone or in combination with one or more deletions and/or substitutions.
  • the MIC-1 analogues of the invention include insertions of one or more unnatural amino acids and/or non-amino acids into the sequence of MIC-1.
  • protein or "polypeptide”, as e.g. used herein, refers to a compound which comprises a series of amino acids interconnected by amide (or peptide) bonds.
  • Amino acids are molecules containing an amine group and a carboxylic acid group, and, optionally, one or more additional groups, often referred to as a side chain.
  • amino acid includes coded (or proteinogenic or natural) amino acids
  • Non-coded amino acids are those which are naturally incorporated into proteins.
  • the standard amino acids are those encoded by the genetic code.
  • Non-coded amino acids are either not found in proteins, or not produced by standard cellular machinery (e.g., they may have been subject to post-translational modification).
  • all amino acids of the MIC-1 proteins for which the optical isomer is not stated is to be understood to mean the L-isomer (unless otherwise specified).
  • amino acid residues may be identified by their full name, their one-letter code, and/or their three-letter code. These three ways are fully equivalent.
  • amino acid codes are provided below:
  • Glycine G and Gly; Proline: P and Pro; Alanine: A and Ala; Valine: V and Val; Leucine: L and Leu; Isoleucine: I and He; Methionine: M and Met; Cysteine: C and Cys; Phenylalanine: F and Phe; Tyrosine: Y and Tyr; Tryptophan : W and Trp; Histidine: H and His; Lysine: K and Lys; Arginine: R and Arg; Glutamine: Q and Gin; Asparagine: N and Asn; Glutamic Acid : E and Glu; Aspartic Acid : D and Asp; Serine: S and Ser; and
  • Threonine T and Thr. N-terminal amino acid extension
  • the MIC-1 compounds of the invention comprise an N-terminal amino acid extension.
  • N-terminal amino acid extension means that the N- terminal of the MIC-1 polypeptide is attached to the C-terminal of the N-terminal amino acid extension via a peptide bond.
  • N-terminal amino acid extension means the same thing and are used interchangeably.
  • the compound of the invention comprises human MIC-1 monomer sequence (SEQ ID NO : l) with an amino acid extension attached at the N-terminal, i.e. the Alanine at positon 1 (Al) via a peptide bond.
  • the N-terminal amino acid extension is up to 200 amino acid residues long. In a particular embodiment of the invention the N- terminal amino acid extension has from 3 to 36 amino acid residues.
  • the N-terminal amino acid extension has a surplus of acidic amino acid residues (Aspartic acid and/or Glutamic acid) of at least 3, 4, 5 or 6 compared to the number of basic amino acid residues (Lysine, Arginine and/or Histidine).
  • a "surplus" of acidic amino acid residues means that the number of acidic residues exceeds the number of basic residues.
  • a defined value of the surplus of acidic amino acid residues is calculated as the number of acidic residues minus the number of basic residues.
  • Methionine is the initial amino acid for protein expression in prokaryotic cells (e.g. bacteria, for instance, E.coli).
  • the initial Methionine is removed from the protein during the protein expression. Therefore, the initial Methionine is not included in the sequence of the N-extension of MIC-1 compound.
  • the start codon, coding the initial Methionine is required for the protein translation initiation and should be incorporated right in front of the nucleotide sequence for protein expression without exception.
  • the MIC-1 compounds of the invention comprise a protractor.
  • the protractor is covalently attached to a specific amino acid residue of the MIC-1 polypeptide or N- terminal amino acid extension.
  • protractors relates to the properties of conveying extended plasma exposure ("half-life extending moiety") and is herein understood to refer to a chemical group attached to an amino acid site chain functionalities such as -SH, -OH, -COOH, - CONH2, -NH2 that can increase in vivo circulatory half-life of MIC-1 when conjugated to the MIC-1.
  • protractors include but are not limited to : fatty acids and derivatives thereof, Hydroxy Alkyl Starch (HAS) e.g.
  • HES Hydroxy Ethyl Starch
  • PEG Poly Ethylen Glycol
  • HAP Poly (Glyx-Sery)n
  • HAP Hyaluronic acid
  • HEP Heparosan polymers
  • PC polymer Phosphorylcholine-based polymers
  • Fleximers Dextran
  • Poly-sialic acids PSA
  • Fc domain Transferrin
  • Albumin Elastin like peptides
  • unstructured and repeated amino sequences e.g. XTEN polymers
  • Albumin binding peptides e.g. XTEN polymers
  • the protractor is capable of forming non- covalent associations with albumin, thereby increasing the blood/plasma exposure time of the MIC-1 compound, and also having the effect of protracting the time of action of the MIC-1 compound, due to the fact that the association of the MIC-1 compound and albumin is only slowly disintegrated to release the active pharmaceutical ingredient.
  • the fatty acid comprising protractors of the invention are capable of forming non-covalent associations with albumin and thereby prolonging plasma half-life of the MIC-1 compound compared to human wide type MIC-1.
  • the protractor is covalently attached to a cysteine residue of the N-terminal amino acid extension of the MIC-1 polypeptide.
  • the protractor comprises a haloacetamide group, which reacts with the thiol group of a cysteine residue, under formation of a covalent sufur-carbon bond (this process being referred to as Cys-alkylation) which is also referred to as a thio-ether bond.
  • the protractor comprises a maleimide group, which reacts with the thiol group of a cysteine residue, under formation of a covalent sulfur-carbon bond.
  • the protractor is covalently attached the N-terminal amino acid of the N-terminal amino acid extension.
  • Fatty acid comprising protractor
  • the protractor comprises, or consists of, at least one of each of Chem. 1, Chem. 2, Chem. 3, and Chem. 4: wherein Chem. 1 is selected from the group consisting of:
  • Chem. 1A HOOC-(CH 2 ) x -CO-*
  • Chem. 1C HOOC-benzene-0-(CH 2 ) y -CO-*
  • x is an integer in the range of 12-20
  • Chem.2 is selected from the group consisting of:
  • Chem.2A *-NH-CH(COOH)-(CH 2 ) m -CO-*
  • m of Chem.2 is an integer in the range of 1-5; wherein Chem.3 is
  • k of Chem.3 is an integer in the range of 1-10
  • n is an integer in the range of 1-5; and wherein Chem.4 is selected from
  • n is an integer in range of 2-6 or
  • Chem.4 is selected from the group consisting of Formula I, II or III
  • Chem. 1, Chem. 2, Chem. 3, and Chem. 4 are interconnected via amide bonds, connecting the*-NH end of a Chem. to the CO-* end of another Chem..
  • the protractor of the invention comprises one Chem. 1, one Chem. 4, and one or more of Chem. 2 and Chem. 3.
  • the protractor consists of one Chem. 1 element, two Chem. 2 elements, two Chem. 3 elements, and one Chem. 4 element.
  • Chem. 2 and Chem. 3 both hold a -NH- and CO- end allowing them to be linked by amide bonds to each other and to either -CO- or -NH- of Chem. 1 or Chem. 4.
  • Chem. 4 has a -NH- end (capable of forming an amide bond with Chem. 2 or Chem. 3).
  • a -NH-CO-CH 2 - end which in the unreacted form is a haloacetamide capable of reacting with the thiol group of a cysteine residue, or a (-N*- CO-CH2-CH**-CO)-end, the parenthesis representing a cyclic structure, which in the unreacted form is a maleimide capable of reacting with the thiol group of the cysteine; or an aldehyde capable of reacting with the N-terminal amino group in a reductive alkylation reaction.
  • the length of the carbon chain of Chem. l defined by x or y may vary from 12-20 for x and 5-15 for y. Shorter or longer versions may be favoured for different types of protractors.
  • *-(CH 2 ) x -* refers to straight alkylene in which x is an integer in the range of 12-20, such as 14-18 or such as 16.
  • This Chem. 1 may be briefly referred to as C18 diacid, i.e. a fatty di-carboxylic acid with 18 carbon atoms.
  • x 16 the structure of this linker element corresponds to Chem. la : HOOC-(CH 2 ) 16 -CO-*.
  • Chem. 1 is selected from the group consisting of:
  • Chem. lc HOOC-benzene-0-(CH 2 ) 9 -CO-*
  • Chem. Id (lH-tetrazol-5-yl)-(CH 2 ) 15 -CO-*
  • Chem. 2 is selected from the group consisting of:
  • Chem. 2c *-NH-CH 2 -cyclohexane-CO-*.
  • protractor is attached to a Cysteine residue; and the protractor comprises, or consists of, at least one of each of Chem. 1, Chem. 2, Chem. 3 and Chem. 4;
  • Chem. 1 is selected from the group consisting of:
  • Chem. 1A HOOC-(CH 2 ) x -CO-*
  • Chem. 1C HOOC-benzene-0-(CH 2 ) y -CO-*
  • x is an integer in the range of 12-20
  • Chem. 2 is selected from the group consisting of:
  • Chem. 2A *-(NH-CH(COOH)-(CH 2 ) m -CO) k *,
  • Chem. 2C *-(NH-(CH 2 ) m -cyclohexane-CO) k -*,
  • n of Chem. 2 is an integer in the range of 1-5, and
  • k is an integer in the range of 0-4; wherein Chem. 3 is
  • k of Chem. 3 is an integer in the range of 1-10,
  • n is an integer in the range of 1-5, and
  • Chem. 4A *-NH-(CH 2 ) m -NH-CO-CH 2 -*
  • Chem. 4B *-NH-CH(COOH)-(CH 2 ) m -NH-CO-CH 2 -*,
  • m of Chem. 4 is an integer in the range of 1-5; and wherein Chem. 1, Chem. 2, Chem. 3, and Chem. 4 are interconnected via amide bonds, connecting the*-NH end of a Chem. to the CO-* end of another Chem., and wherein the CH 2 -* end of Chem. 4 is connected to the sulphur atom of the Cysteine residue of the amino acid extension.
  • the protractor is attached to an N-terminal amino acid, and comprises, or consists of, at least one of each of Chem. 1, Chem. 2, Chem. 3 and Chem. 4;
  • Chem. 1 is selected from the group consisting of:
  • x is an integer in the range of 12-20
  • Chem. 2 is selected from the group consisting of:
  • Chem. 2A *-(NH-CH(COOH)-(CH 2 ) m -CO) k *,
  • Chem. 2C *-(NH-(CH 2 ) m -cyclohexane-CO) k -*,
  • n of Chem. 2 is an integer in the range of 1-5, and
  • k is an integer in the range of 0-4; wherein Chem. 3 is
  • k of Chem. 3 is an integer in the range of 1-10,
  • n is an integer in the range of 1-5, and
  • I is an integer in the range of 0-5; elected from the group consisting of Formula I, II or III :
  • Chem. 1, Chem. 2, Chem. 3, and Chem. 4 are interconnected via amide bonds, connecting the*-NH end of a Chem. to the CO-* end of another Chem., and wherein the CH 2 -* end of Chem. 4 is connected to the alpha-amino group of the N- terminal of the amino acid extension.
  • the compounds/protractors of the invention may exist in different stereo- isomeric forms having the same molecular formula and sequence of bonded atoms, but differing only in the three-dimensional orientation of their atoms in space.
  • the stereoisomerism of the exemplified compounds/protractors of the invention is indicated in the experimental section, in the names as well as the structures, using standard nomenclature. Unless otherwise stated the invention relates to all stereoisomeric forms of the claimed compounds/protractors. Isoelectric point (pi)
  • the calculated pi of the MIC-1 polypeptide with an N-terminal amino acid extension is defined as the pH at which the net calculated charge of the MIC-1 polypeptide with the N-extension is zero.
  • the calculated charge of the MIC-1 polypeptide with the N-extension as a function of pH is obtained using the pKa values of the amino acid residues described in Table 1 and the method described by B. Skoog and A.
  • Table 1 pKa of amino acid residues used for calculating pi.
  • the pKa values are those described in "Correlation of Electrophoretic Mobilities from Capillary Electrophoresis with Physicochemical Properties of Proteins and Peptides by Rickard EC, Strohl MM, Nielsen RG. Analytical Biochemistry 1991, vol 197, pp 197-207".
  • the MIC-1 compounds of the invention have good biophysical properties.
  • the MIC-1 compounds of the invention are biologically active. For example they are potent, binds to and activate the MIC-1 receptor complex. Also MIC-1 compounds exhibit protracted plasma exposure defined as longer half-life. For example MIC-1 compounds have a markedly longer plasma half-life when administered i.v. to rat and/or mini pigs compared to MIC-1 (SEQ ID 1). The particular combination of retained receptor potency and long plasma half-life may be highly desirable.
  • the compounds of the invention have retained MIC-1 receptor potency relative to human MIC-1 (SEQ ID NO : l).
  • Receptor potency and efficacy can be measured in mammalian cells transfected with human MIC-1 receptor (hGFRAL, GDNF family receptor alpha like) and its signalling co-receptor hRET (proto-oncogene tyrosine- protein kinase receptor Ret ).
  • MIC-1 compounds activation of the receptor complex is measured by phosphorylation of extracellular signal-regulated kinases (ERKs) as described in Example 6.
  • ERKs extracellular signal-regulated kinases
  • receptor potency and efficacy is measured on MIC-1 compounds as homodimers.
  • the compounds of the invention are potent in vivo, which may be determined as is known in the art in any suitable animal model.
  • the non-obese Sprague Dawley rat is one example of a suitable animal model, and the changes in food intake may be determined in such rats in vivo, e.g. as described in Example 14.
  • the compounds of the invention inhibits in vivo food intake in non-obese Sprague Dawley rats.
  • the MIC-1 compounds of the invention are protracted and have an extended in vivo plasma half-life, which can be determined in a suitable pharmacokinetic in vivo study.
  • Extended plasma exposure may be determined as plasma half-life (TV2) after i.v. administration to animals such as rats or mini pigs.
  • TV2 plasma half-life
  • the MIC-1 compounds of the invention have a plasma half-life after i.v. administration to rat of at least 10 hour, more preferably between 25- 50 hours, or most preferably at least 50 hours, determined as described in Example 16.
  • the MIC-1 compounds of the invention have a plasma half-life after i.v. administration to mini pigs of at least 50 hours, more preferably between 50-200 hours, even more preferably at least 200 hours or most preferably at least 300 hours, determined as described in Example 17.
  • the compounds of the invention are protracted and at the same time retain in vivo potency.
  • the particular combination of retained potency and long plasma half-life may be highly desirable.
  • the human wild type MIC-1 is a hydrophobic protein, with a calculated pi 8.8 based on the homodimer. Consequently, wild type MIC-1 can only be solubilized to around 0.5 mg/ml in neutral pH aqueous buffer systems.
  • the low solubility of MIC-1 significantly hampers its pharmaceutical formulation properties and therapeutic use, so developing a MIC-1 compound with improved solubility would greatly improve the therapeutic utility.
  • the MIC-1 polypeptides with an N-extension of the invention have improved solubility (i.e. are more soluble) relative to human MIC-1 of SEQ ID NO: 1.
  • solubility is measured as described in Example 4.
  • the MIC-1 polypeptides with an N-extension of the invention have a solubility of at least 1 mg/ml in Tris buffer at pH 8.0. In other embodiments, the MIC-1 polypeptides with an N-extension of the invention have a solubility of at least 5 mg/ml, at least 10 mg/ml, at least 30 mg/ml, or at least 40 mg/ml in Tris buffer at pH 8.0.
  • solubility is measured on MIC-1 compounds and MIC-1 polypeptides with an N-extension as homodimers.
  • the human wild type MIC-1 sequence is chemically unstable and several residues of the amino acid sequence could be modified during storage, including deamidation on Asparagine at position 3 (N3) and oxidation of methionines M43, M57 and M86. Chemical instability of certain residues could impact pharmaceutical properties so developing chemical stable MIC-1 compounds would be another important part of making a MIC-1 therapeutic compound.
  • the compounds of the invention have improved chemical stability relative to human MIC-1 of SEQ ID NO : l .
  • chemical stability refers to chemical changes in the polypeptide structure leading to formation of chemical degradation products potentially having a reduced biological activity, decreased solubility, and/or increased immunogenic effect as compared to the intact polypeptide.
  • the chemical stability can be evaluated by measuring the amount of chemical degradation products at various time-points after exposure to different environmental conditions, e.g. by SEC-HPLC, and/or RP-HPLC.
  • certain residues of the MIC-1 monomer sequence (SEQ ID NO : l) is modified, e.g. by substitution to increase the chemical stability of the MIC-1 compounds.
  • N3 is deleted or substituted with other amino acids, e.g. E or Q.
  • Methionine is substituted with other amino acids, e.g. E or L.
  • the MIC-1 compounds of the invention have low immunogenicity risk.
  • MIC-1 polypeptides with an N-terminal amino acid extension of the present invention may be produced by means of recombinant protein technology known to persons skilled in the art.
  • nucleic acid sequences encoding the proteins of interest or functional variants thereof are modified to encode the desired MIC-1 polypeptide with an N-extension. This modified sequence is then inserted into an expression vector, which is in turn transformed or transfected into the expression host cells.
  • the nucleic acid construct encoding the MIC-1 polypeptide with an N-extension may suitably be of genomic, cDNA or synthetic origin. Amino acid sequence alterations are accomplished by modification of the genetic code by well-known techniques.
  • the DNA sequence encoding the MIC-1 polypeptide with an N-extension is usually inserted into a recombinant vector which may be any vector, which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e. a vector, which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
  • the vector is preferably an expression vector in which the DNA sequence encoding the MIC-1 polypeptide with an N-extension is operably linked to additional segments required for transcription of the DNA.
  • operably linked indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence coding for the polypeptide until it terminates within a terminator.
  • expression vectors for use in expressing the MIC-1 polypeptide with an N- extension will comprise a promoter capable of initiating and directing the transcription of a cloned gene or cDNA.
  • the promoter may be any DNA sequence, which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • N-extension will also comprise a terminator sequence, a sequence recognized by a host cell to terminate transcription.
  • the terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.
  • Expression of the MIC-1 polypeptide with an N-extension can be aimed for either intracellular expression in the cytosol of the host cell or be directed into the secretory pathway for extracellular expression into the growth medium.
  • Intracellular expression is the default pathway and requires an expression vector with a DNA sequence comprising a promoter followed by the DNA sequence encoding the MIC-1 polypeptide with an N-extension followed by a terminator.
  • a secretory signal sequence (also known as signal peptide or a pre sequence) is needed as an extension of the MIC-1 sequence.
  • a DNA sequence encoding the signal peptide is joined to the 5' end of the DNA sequence encoding the MIC-1 polypeptide with an N-extension in the correct reading frame.
  • the signal peptide may be that normally associated with the protein or may be from a gene encoding another secreted protein.
  • the host cell into which the DNA sequence encoding the MIC-1 polypeptide with an N-extension is introduced may be any cell that is capable of expressing the MIC-1 polypeptide with an N-extension either intracellular ⁇ or extracellularly.
  • the MIC-1 polypeptide with an N-extension may be produced by culturing a host cell containing a DNA sequence encoding the MIC-1 polypeptide with an N-extension and capable of expressing the MIC-1 polypeptide with an N-extension in a suitable nutrient medium under conditions permitting the expression of the MIC-1 polypeptide with an N-extension.
  • Non-limiting examples of host cells suitable for expression of MIC-1 polypeptide with N- extension are: Escherichia coli, Saccharomyces cerevisiae, as well as human embryonic kidney (HEK), Baby Hamster Kidney (BHK) or Chinese hamster ovary (CHO) cell lines. If posttranslational modifications are needed, suitable host cells include yeast, fungi, insects and higher eukaryotic cells such as mammalian cells.
  • MIC-1 polypeptide with an N-extension may be recovered and purified to the required quality by conventional techniques.
  • Non-limiting examples of such conventional recovery and purification techniques are centrifugation, solubilization, filtration, precipitation, ion-exchange chromatography, immobilized metal affinity chromatography (IMAC), Reversed phase - High Performance Liquid Chromatography (RP-HPLC), gel-filtration and freeze drying.
  • MIC-1 proteins examples include Chich et al, Anal. Biochem, 1995, 224, pp 245-249 and Xin et al., Protein Expr. Purif. 2002, 24, pp530-538.
  • MIC-1 polypeptides with an N-terminal amino acid extension can be expressed in bacteria such as E. coli.
  • large scale protein production of the MIC-1 polypeptides with an N-extension could take of using Inclusion Bodies (IB) as this represent an advantageous approach to controlling process recovery, protein purity, protease degradation and general protein stability. This becomes particular important for large scale protein production.
  • IB Inclusion Bodies
  • the balance of MIC-1 polypeptides with an N-extension solubility partly controlled by the calculated pi and IB formation.
  • the route of administration may be any route which effectively transports a compound of this invention to the desired or appropriate place in the body, such as parenterally, for example, subcutaneously, intramuscularly or intraveneously.
  • a compound of this invention can be administered orally, pulmonary, rectally, transdermal ⁇ , buccally, sublingually, or nasally.
  • compositions comprising a compound of the invention or a pharmaceutically acceptable salt, amide, or ester thereof, and a pharmaceutically acceptable excipient may be prepared as is known in the art.
  • excipient broadly refers to any component other than the active therapeutic ingredient(s).
  • the excipient may be an inert substance, an inactive substance, and/or a not medicinally active substance.
  • the excipient may serve various purposes, e.g. as a carrier, vehicle, diluent, tablet aid, and/or to improve administration, and/or absorption of the active substance.
  • the treatment with a compound according to the present invention may also be combined with one or more pharmacologically active substances, e.g., selected from antiobesity agents, appetite regulating agents, and agents for the treatment and/or prevention of complications and disorders resulting from or associated with obesity.
  • one or more pharmacologically active substances e.g., selected from antiobesity agents, appetite regulating agents, and agents for the treatment and/or prevention of complications and disorders resulting from or associated with obesity.
  • the present invention relates to a compound of the invention, for use as a medicament.
  • the compound of the invention may be used for the following medical treatments:
  • the invention relates to a method for reduction of appetite. In some embodiments the invention relates to a method for reduction of food intake.
  • the invention relates to a method for treatment or prevention of obesity. In some embodiments the invention relates to use of the MIC-1 compounds of the invention for treatment or prevention of obesity. In some
  • the subject suffering from obesity is human, such as an adult human or a paediatric human (including infants, children, and adolescents).
  • a human subject suffering from obesity has a BMI of >30; this subject may also be referred to as obese.
  • the human subject suffering from obesity has a BMI of >35 or a BMI in the range of >30 to ⁇ 40.
  • the obesity is severe obesity or morbid obesity, wherein the human subject has a BMI of >40.
  • the invention relates to a method for treatment or prevention of overweight, optionally in the presence of at least one weight-related comorbidity. In some embodiments the invention relates to use of the MIC-1 compounds of the invention for treatment or prevention of overweight, optionally in the presence of at least one weight-related comorbidity.
  • the subject suffering from overweight is human, such as an adult human or a paediatric human (including infants, children, and adolescents).
  • a human subject suffering from overweight has a BMI of >25, such as a BMI of >27.
  • a human subject suffering from overweight has a BMI in the range of 25 to ⁇ 30 or in the range of 27 to ⁇ 30.
  • the weight-related comorbidity is selected from the group consisting of hypertension, diabetes (such as type 2 diabetes), dyslipidaemia, high cholesterol, and obstructive sleep apnoea.
  • the invention relates to a method for reduction of body weight. In some embodiments the invention relates to use of the MIC-1 compounds of the invention for reduction of body weight.
  • a human to be subjected to reduction of body weight according to the present invention has a BMI of >25, such as a BMI of >27 or a BMI of >30. In some embodiments the human to be subjected to reduction of body weight according to the present invention has a BMI of >35 or a BMI of >40.
  • the invention relates to a method for treatment or prevention of cardiovascular diseases like arteriosclerosis and other disorders such as steatohepatitis, and diabetic nephropathy.
  • a MIC-1 polypeptide means one MIC-1 polypeptide or more than one MIC-1 polypeptide.
  • a MIC-1 compound comprising a MIC-1 polypeptide with an N-terminal amino acid extension and a protractor, wherein the protractor is attached to the amino acid extension.
  • Chem. 1A HOOC-(CH 2 ) x -CO-*
  • Chem. 1C HOOC-benzene-0-(CH 2 ) y -CO-*
  • x is an integer in the range of 12-20
  • Chem. 2 is selected from the group consisting of:
  • Chem. 2A *-(NH-CH(COOH)-(CH 2 ) m -CO) k *,
  • Chem. 2C *-(NH-(CH 2 ) m -cyclohexane-CO) k -*,
  • n of Chem. 2 is an integer in the range of 1-5, and
  • k is an integer in the range of 0-4; wherein Chem. 3 is
  • k of Chem. 3 is an integer in the range of 1-10,
  • n is an integer in the range of 1-5, and
  • Chem. 4A *-NH-(CH 2 ) m -NH-CO-CH 2 -*
  • Chem. 4B *-NH-CH(COOH)-(CH 2 ) m -NH-CO-CH 2 -*,
  • Chem. 4C *-NH-(CH2)m-CH(COOH)-NH-CO-CH2-*, and ⁇ NH-(CH2)n-NH-CO-(CH2)m
  • m of Chem. 4 is an integer in the range of 1-5 and n is an integer in the range of 2-6; and wherein Chem. 1, Chem. 2, Chem. 3, and Chem. 4 are interconnected via amide bonds, connecting the*-NH end of a Chem. to the CO-* end of another Chem., and wherein the CH 2 -* end of Chem. 4 is connected to a sulfur atom of the Cysteine residue of the amino acid extension. 5.
  • x is an integer in the range of 12-20
  • Chem. 2 is selected from the group consisting of:
  • Chem. 2A *-(NH-CH(COOH)-(CH 2 ) m -CO) k *,
  • Chem. 2C *-(NH-(CH 2 ) m -cyclohexane-CO) k -*,
  • n of Chem. 2 is an integer in the range of 1-5, and
  • k is an integer in the range of 0-4; wherein Chem. 3 is
  • k of Chem. 3 is an integer in the range of 1-10, n is an integer in the range of 1-5, and
  • I is an integer in the range of 0-5; elected from the group consisting of Formula I, II or III :
  • Chem. 2c *-NH-CH 2 -cyclohexane-CO-*.
  • n is of 0-3 and m is 0-3
  • Chem. 4a *-NH-(CH 2 ) 2 -NH-CO-CH 2 -* and
  • Chem. 4b *-NH-CH(COOH)-(CH 2 ) 4 -NH-CO-CH 2 -*.
  • n is 1-4.
  • HAS Hydroxy Alkyl Starch
  • HAS Hydroxy Ethyl Starch
  • PEG Poly Ethylen Glycol
  • HAP Poly (Glyx-Sery)n
  • HAP Hyaluronic acid
  • HEP Heparosan polymers
  • PC polymer Phosphorylcholine-based polymers
  • Fleximers Dextran
  • Poly-sialic acids PSA
  • Fc domain Transferrin, Albumin, Elastin like peptides, XTEN polymers, Albumin binding peptides, a CTP peptide, and any combination thereof.
  • MIC-1 compound according to any of the preceding embodiments, wherein the amino acid extension is in the range of 3-100, 3-50, 3-40, 3-30, 5-100, 5-50, 5-40, 5- 30, 10-100, 10-50, 10-40, 10-30, 3-36, 3-30, 3-25, 3-24, 3-12, 4-36, 4-30, , 4-24, 4- 12, 5-36, 5-30, 5-24, 5-12, 6-36, 6-30, 6-24, 6-12, 7-36, , 7-30, 7-24, 7-12, 8-36, 8- 30, 8-24, 8-12, 30-36, 32-36, 30-34, or 30-32 amino acid residues in length.
  • the MIC-1 compound according to any of the preceding embodiments, wherein the amino acid extension is in the range of 3-36 or 30-32 amino acid residues in length.
  • the amino acid extension has a surplus of acidic amino acid residues (Aspartic acid or Glutamic acid) of at least 3, 4, 5, 6, 7, 8, 9 or 10 compared to the number of basic amino acid residues (Lysine, Arginine or Histidine) . 30.
  • amino acid extension is composed of amino acid residues selected among the group consisting of A, C, E, G, P, S, T, Q, N and D and wherein the amino acid extension comprises at least three E and/or D amino acid residues.
  • amino acid extension comprises two of sequences selected from the group consisting of SPAGSPTSTEEG, TSESATPESGPG, TSTEPSEGSAPG and SEPATSGSETPG, and wherein one of the amino acid of the amino acid extension is replaced with Cysteine.
  • amino acid extension further comprises 6-8 consecutive amino acids of SPAGSPTSTEEG, TSESATPESGPG, TSTEPSEGSAPG or SEPATSGSETPG, such as the first 6-8 amino acid residues, the last 6- 8 residues or the internal 6-8 residues, and wherein one of the amino acid of the amino acid extension is replaced with Cysteine.
  • Cysteine locates in any position of the N-terminal amino acid extension.
  • MIC-1 polypeptide is at least 1, 3, 5, 10, 15, 19, 23, 26, 29, or 32 amino acids (including Cysteine residue of the N-terminal extension, but not including N-terminal amino acid of MIC-1 polypeptide). 42.
  • the MIC-1 compound according to embodiment 40, wherein the distance between Cysteine residue of the N-terminal extension and the N-terminal amino acid of the MIC-1 polypeptide is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 amino acids (including Cysteine residue of the N-terminal extension, but not including N-terminal amino acid of MIC-1 polypeptide).
  • SEPCTSGSETPGTSESATPESGPGTSTEPSEG (SEQ ID NO : 225),
  • SEPCTSGSETPGTSESATPESGPGTSTEPS (SEQ ID NO : 240),
  • SEPCTSGSETPGTSESATPESGPG SEQ ID NO : 242
  • SEPCTSGSETPGTSESATPES SEQ ID NO : 243
  • SEPCTSGSETPG (SEQ ID NO : 244)
  • SEPCTSGSETPGSPAGSPTSTEEGSPAGSP (SEQ ID NO : 245), SEPCTSGSETPGTSESATPESGPGSPAGSP (SEQ ID NO : 246), SEPCTSGSETPGTSTEPESGSAPGSPAGSP (SEQ ID NO : 247), SEPCTSGSETPGSPAGSPTSTEEGTSESAT (SEQ ID NO : 248), S E PCTSG S ETPGTS E S ATP E S G PGTS E S AT (SEQ ID NO : 249), S E PCTSGS ETPGTSTE PE SG S APGTS E SAT (SEQ ID NO : 250), S E PCTSGS ETPGS PAG S PTSTE EGTSTE PE (SEQ ID NO : 251), S E PCTSGS ETPGTS E S ATPE SG PGTSTE PE (SEQ ID NO : 252), SEPCTSGSETPGTSTEPESGSAPGTSTEPE (SEQ ID NO : 253), S E PCTSGS ETPGS PAG S PTSTE
  • SEPCTSGSETPGSPAGSPTSTEEGTSESATPESGPG (SEQ ID NO : 269)
  • SEPCTSGSETPGSPAGSPTSTEEGSPAGSPTSTEEG (SEQ ID NO : 270)
  • SEPCTSGSETPGTSESATPESGPGSPAGSPTSTEEG (SEQ ID NO : 272)
  • SEPCTSGSETPGTSESATPESGPGTSESATPESGPG (SEQ ID NO : 273)
  • SEPCTSGSETPGTSESATPESGPGTSTEPESGSAPG SEQ ID NO : 274.
  • SEPCTSGSETPGTSESATPESGPGSEPATSGSETPG (SEQ ID NO : 275)
  • SEPCTSGSETPGTSTEPESGSAPGSPAGSPTSTEEG (SEQ ID NO : 276)
  • SEPCTSGSETPGTSTEPESGSAPGTSESATPESGPG (SEQ ID NO : 277)
  • SEPCTSGSETPGTSTEPESGSAPGTSTEPESGSAPG (SEQ ID NO : 278)
  • SEPCTSGSETPGTSTEPESGSAPGSEPATSGSETPG SEQ ID NO : 279
  • SEPCTSGSETPGSEPATSGSETPGSPAGSPTSTEEG SEQ ID NO : 280
  • SEPCTSGSETPGSEPATSGSETPGTSESATPESGPG SEQ ID NO : 281)
  • SEPCTSGSETPGSEPATSGSETPGTSTEPESGSAPG SEQ ID NO : 282
  • SEPCTSGSETPGSEPATSGSETPGSEPATSGSETPG (SEQ ID NO : 283)
  • GECPGEQPGEQPGEQPGEQPGEQP (SEQ ID NO : 285)
  • SEPACSGSETPGTSESATPESGPGTSTEPSEG (SEQ ID NO : 229)
  • amino acid extension is composed of amino acid residues selected among the group consisting of A, E, G, P, S, T, Q and D, wherein the amino acid extension comprises at least three E and/or D amino acid residues.
  • the MIC-1 compound according to embodiment 48 or 49, wherein the amino acid extension comprises at least three E and at least one P.
  • the MIC-1 compound according to embodiment 50, wherein the amino acid extension further comprises S, G, T and A. 52.
  • the MIC-1 compound according to embodiment 51, wherein the amino acid extension comprises 6 Ser, 4 Pro, 4 Gly, 4 Thr, 4 Glu and 2 Ala.
  • the MIC-1 compound according to embodiment 51, wherein the amino acid extension comprises two of sequences selected from the group consisting of SPAGSPTSTEEG, TSESATPESGPG, TSTEPSEGSAPG and SEPATSGSETPG.
  • MIC-1 compound according to embodiment 53 wherein the amino acid extension further comprises 6-8 consecutive amino acids of SPAGSPTSTEEG, TSESATPESGPG,
  • TSTEPSEGSAPG or SEPATSGSETPG such as the first 6-8 amino acid residues, the last 6- 8 residues or the internal 6-8 residues.
  • the amino acid extension comprises one or more of the following sequences SPAGSP (SEQ ID NO:4), TSESAT (SEQ ID NO : 5), TSTEPE (SEQ ID NO : 6), SEPATS (SEQ ID NO : 7), TSTEEG (SEQ ID NO: 8), PESGPG (SEQ ID NO:9), SGSAPG (SEQ ID NO: 10), GSETPG (SEQ ID NO: 11), SEPATSGSETPGSPAGSPTSTEEG (SEQ ID NO : 12),
  • SPAGSP SEQ ID NO:4
  • TSESAT SEQ ID NO : 5
  • TSTEPE SEQ ID NO : 6
  • SEPATS SEQ ID NO : 7
  • TSTEEG SEQ ID NO: 8
  • PESGPG SEQ ID NO:9
  • SGSAPG SEQ ID NO: 10
  • GSETPG SEQ ID NO: 11
  • SEPATSGSETPGSPAGSPTSTEEG SEQ ID NO : 12
  • SEPATSGSETPGTSESATPESGPG SEQ ID NO : 13
  • SEPATSGSETPGTSTEPESGSAPG SEQ ID NO: 14
  • GEPS (SEQ ID NO: 118), GPSE (SEQ ID NO: 119), GPES (SEQ ID NO: 120), GSPE (SEQ ID NO: 121), GSEP (SEQ ID NO: 122), GEPQ (SEQ ID NO: 123), GEQP (SEQ ID NO: 124), GPEQ (SEQ ID NO: 125), GPQE (SEQ ID NO: 126), GQEP (SEQ ID NO:127)or GQPE (SEQ ID NO: 128), PEDEETPEQE (SEQ ID NO: 129), PDEGTEEETE (SEQ ID NO: 130),
  • PAAEEEDDPD (SEQ ID NO: 131), AEPDEDPQSED (SEQ ID NO: 132), AEPDEDPQSE (SEQ ID NO : 133), AEPEEQEED (SEQ ID NO : 134), AEPEEQEE (SEQ ID NO : 135), GGGS (SEQ ID NO : 136), GSGS (SEQ ID NO : 137), GGSS (SEQ ID NO : 138) and SSSG (SEQ ID NO : 139) .
  • MIC- 1 compound according to any one of embodiments 1-2 and 5-33, wherein the amino acid extension comprises one or more of the following sequences SPAGSP, TSESAT, TSTEPE, SEPATS, TSTEEG, PESGPG, SGSAPG, GSETPG,
  • SEPATSGSETPGTSESATPESGPGSPAGSP SEPATSGSETPGTSTEPESGSAPGSPAGSP, S E PATSG S ETPG S PAGS PTSTE EGTS ES AT, S E PATS G S ETPGTS E S ATP E SG PGTS E SAT, SEPATSGSETPGTSTEPESGSAPGTSESAT, SEPATSGSETPGSPAGSPTSTEEGTSTEPE, SEPATSGSETPGTSESATPESGPGTSTEPE, SEPATSGSETPGTSTEPESGSAPGTSTEPE, S E P ATSG S ETPGSPAGSPTSTEEGSE PATS , SEPATSGSETPGTSESATPESGPGSEPATS, SEPATSGSETPGTSTEPESGSAPGSEPATS, SEPATSGSETPGSPAGSPTSTEEGTSTEEG, SEPATSGSETPGTSESATPESGPGTSTEEG, SEPATSGSETPGTSTEPESGSAPGTSTEEG, SEPATSGSETPGSPAGSPTSTEEGPESGPG, SEPATSGSETPGTS
  • GEPQ GEPS, GGGS, GSGS, GGSS, and SSSG.
  • the amino acid extension comprises any combination of any 2-6 of the following sequences SPAGSP, TSESAT, TSTEPE, SEPATS, TSTEEG, PESGPG, SGSAPG, GSETPG, GEPQ, GEPS, GGGS, GSGS, GGSS, and SSSG.
  • MIC-1 compound according to any one of embodiments 1-2 and 5-33, wherein the amino acid extension comprises one or more of the following sequences GEPS, GPSE, GPES, GSPE, GSEP, GEPQ, GEQP, GPEQ, GPQE, GQEP, GQPE, GGGS, GSGS, GGSS, and SSSG.
  • the MIC-1 compound according to embodiment 61, wherein the amino acid extension comprises any combination of 2-9 of the following sequences GEPS, GPSE, GPES, GSPE, GSEP, GEPQ, GEQP, GPEQ, GPQE, GQEP, GQPE, GGGS, GSGS, GGSS, and SSSG.
  • the amino acid extension comprises one or more of the following sequences GEPS, GPSE, GPES, GSPE, GSEP, GGGS, GSGS, GGSS, and SSSG.
  • MIC-1 compound according to embodiment 63, wherein the amino acid extension comprises any combination of 2-9 of the following sequences GEPS, GPSE, GPES, GSPE,
  • GSEP GGGS, GSGS, GGSS, and SSSG.
  • the MIC-1 compound according to embodiment 64, wherein the amino acid extension comprises one or more of the following sequences GEPSGEPSGEPSGEPSGEPSGEPS (SEQ ID NO : 140), GPSEGPSEGPSEGPSEGPSE (SEQ ID NO: 141), GPESGPESGPESGPESGPES (SEQ ID NO : 142), GSPEGSPEGSPEGSPEGSPE (SEQ ID NO : 143), and
  • GSEPGSEPGSEPGSEPGSEP (SEQ ID NO : 144).
  • MIC-1 compound according to any one of embodiments 1-2 and 5-33, wherein the amino acid extension comprises one or more of the following sequences GEPQ, GEQP,
  • GPEQ GPQE, GQEP, GQPE, GGGS, GSGS, GGSS, and SSSG.
  • the MIC-1 compound according to embodiment 66, wherein the amino acid extension comprises any combination of 2-9 of the following sequences GEPQ, GEQP, GPEQ, GPQE, GQEP, GQPE, GGGS, GSGS, GGSS, and SSSG.
  • the amino acid extension comprises one or more of the following sequences GEPQGEPQGEPQGEPQGEPQGEPQ (SEQ ID NO: 145), GEQPGEQPGEQPGEQPGEQP (SEQ ID NO: 146),
  • PEDEETPEQE PEDEETPEQE, PDEGTEEETE, PAAEEEDDPD, AEPDEDPQSED, AEPDEDPQSE, AEPEEQEED, and AEPEEQEE, GGGS, GSGS, GGSS and SSSG.
  • MIC-1 compound according to any of the embodiments 1-2 and 5-33, wherein the amino acid extension comprises any combination of two to three of the following sequences PEDEETPEQE, PDEGTEEETE, PAAEEEDDPD, AEPDEDPQSED, AEPDEDPQSE, AEPEEQEED, AEPEEQEE and AEEAEEAEEAEEAEE.
  • amino acid extension comprises one or more of the following sequences SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO
  • MIC-1 compound according to any one of preceding embodiments, wherein the amino acid extension comprises 1-4 Glycine and Serine amino acid residues C-terminally.
  • MIC-1 compound according to any of the preceding embodiments, wherein the MIC-1 polypeptide displays at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to wild type MIC-1 (SEQ ID NO : l).
  • MIC-1 compound according to embodiment 70 wherein the MIC-1 polypeptide displays at least 95% sequence identity to wild type MIC-1 (SEQ ID NO: l).
  • MIC-1 compound according to any one of the preceding embodiments wherein the MIC-1 polypeptide comprises one or more of the following substitutions P11E, H 18E, R21E, A30E, M43L, M43E, A47E, R53E, A54E, M57E, M57L, H66E, R67E, L68E, K69E, A75E, A81E, P85E, M86F, M86L, Q90E, T92E, L105E, K107E, K69R, K107R and K91R compared to wild type MIC-1 (SEQ ID NO : l). 81.
  • MIC-1 compound according to any one of the preceding embodiments wherein the MIC-1 polypeptide comprises a H66E substitution compared to MIC-1 of SEQ ID NO : l.
  • MIC-1 compound according to any one of the preceding embodiments wherein the MIC-1 polypeptide comprises a R67E substitution compared to MIC-1 of SEQ ID NO : l.
  • MIC-1 compound according to any one of embodiments 1-75, wherein the MIC-1 polypeptide has a sequence according to SEQ ID NO : 154 (M43L/des-N3).
  • MIC-1 compound according to any one of embodiments 1-75, wherein the MIC-1 polypeptide has a sequence according to SEQ ID NO : 157 (M57L/ ⁇ 1-3).
  • MIC-1 compound according to any one of embodiments 1-75, wherein the MIC-1 polypeptide has a sequence according to SEQ ID NO : l .
  • the MIC-1 compound according to any one of preceding embodiments, wherein the MIC-1 polypeptide and the N-terminal amino acid extension together comprises an amino acid sequence according to SEQ ID NO : 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO : 92, SEQ ID NO : 93, SEQ ID NO : 94, SEQ ID NO : 95, SEQ ID NO : 96, SEQ ID NO : 97, SEQ ID NO : 98, SEQ ID NO : 99, SEQ ID NO: 100, SEQ ID NO : 101, SEQ ID NO : 102, SEQ ID NO: 103, SEQ ID NO : 104, SEQ ID NO : 105, SEQ ID NO : 106, SEQ ID NO : 107, SEQ ID NO: 108, SEQ ID NO : 109, SEQ ID NO : 110, SEQ ID NO : 111, SEQ ID NO : 112, SEQ ID NO: 113
  • SEPCTSGSETPGTSESATPESGPGTSTEPSARGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPR EVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDL LAKDCHCI SEQ ID NO: 301
  • MIC-1 compound according to any one of the preceding embodiments, showing extended plasma half-life compared with MIC-1 of SEQ ID NO : l .
  • MIC-1 compound according to any one of the preceding embodiments, showing improved plasma half-life compared with MIC-1 of SEQ ID NO: l as measured by administrating the compound to rat i.v. and estimate the terminal half-life from changes in plasma concentration of compound over time.
  • the MIC-1 compound according to any one of the preceding embodiments showing maintained potency compared with MIC-1 of SEQ ID NO: l .
  • the MIC-1 compound according to any one of the preceding embodiments showing retained efficacy compared with MIC-1 of SEQ ID NO : l as measured by administrating compound to rat s.c. and measure changes in daily food intake.
  • 104 The MIC-1 compound according to any one of the preceding embodiments, wherein the MIC-1 polypeptide with an amino acid extension has acceptable solubility compared with MIC-1 of SEQ ID NO : l .
  • MIC-1 compound according to any one of embodiments 1-99, wherein the MIC- 1 polypeptide with an amino acid extension has a solubility of 0.5, 1.0, 5.0, 10, 30 or 50 mg/ml as measured at pH 8.0 in a Tris buffer system.
  • MIC-1 compound according to embodiments 1 or 2 wherein the MIC-1 polypeptide comprises a deletion of N3 (des-N3) compared to MIC-1 of SEQ ID NO : l, wherein the amino acid extension has the following sequence
  • MIC-1 compound according to any one of embodiments 1-107 or a pharmaceutically acceptable salt, amide or ester thereof.
  • MIC-1 compound according to any of the preceding embodiments, wherein the compound has improved in vivo efficacy on lowering food intake and/or lowering body weight compared with MIC-1 of SEQ ID NO : l .
  • MIC-1 compound according to any one of embodiments 1-109 and 132-152 for use as a medicament.
  • MIC-1 compound according to any one of embodiments 1-109 and 132-152 for use in the prevention and/or treatment of a metabolic disorder.
  • MIC-1 compound according to embodiment 111 for use in the prevention and/or treatment of a metabolic disorder, wherein the metabolic disorder is obesity, type 2 diabetes, dyslipidemia, or diabetic nephropathy.
  • MIC-1 compound according to any one of embodiments 1-109 and 132-152 for use in the prevention and/or treatment of eating disorders, such as obesity.
  • MIC-1 compound according to embodiment 113 for use in the prevention and/or treatment of obesity by decreasing food intake, reducing body weight, suppressing appetite and/or inducing satiety.
  • MIC-1 compound according to any one of embodiments 1-109 and 132-152 for use in the prevention and/or treatment of a cardiovascular disease.
  • MIC-1 compound according to embodiment 115 for use in the prevention and/or treatment of dyslipidaemia, arteriosclerosis, steatohepatitis, or diabetic nephropathy.
  • composition comprising the MIC-1 compound according to any one of embodiments 1-109 and 132-152 or a pharmaceutically acceptable salt, amide or ester thereof, and one or more pharmaceutically acceptable excipients.
  • a MIC-1 compound according to any one of embodiments 1-109 and 132-152 in the manufacture of a medicament for the prevention and/or treatment of a metabolic disorder, wherein the metabolic disorder is obesity, type 2 diabetes, dyslipidemia, or diabetic nephropathy.
  • a MIC-1 compound according to any one of embodiments 1-109 and 132-152 in the manufacture of a medicament for the prevention and/or treatment of obesity by decreasing food intake, reducing body weight, suppressing appetite and/or inducing satiety.
  • a MIC-1 compound according to any one of embodiments 1-109 and 132-152 in the manufacture of a medicament for the prevention and/or treatment of a cardiovascular disease.
  • a MIC-1 compound according to any one of embodiments 1-109 and 132-152 in the manufacture of a medicament for the prevention and/or treatment of dyslipidaemia, arteriosclerosis, steatohepatitis, or diabetic nephropathy.
  • Method of treating and/or preventing obesity by administering a pharmaceutically active amount of a MIC-1 compound according to any one of embodiments 1-109 and 132-152.
  • Method of treating and/or preventing obesity by decreasing food intake, reducing body weight, suppressing appetite and/or inducing satiety by administering a
  • steatohepatitis or diabetic nephropathy by administering a pharmaceutically active amount of a MIC-1 compound according to any one of embodiments 1-109 and 132-152.
  • Main peak refers to the peak in a purification chromatogram which has the highest UV intensity in milliabsorbance units and which contains the fusion protein.
  • HPLC High performance liquid chromatography
  • SDS-PAGE is Sodium dodecyl sulfate Polyacrylamide gel electrophoresis.
  • IMAC is immobilized metal affinity chromatography.
  • SEC is size exclusion chromatography
  • MS is mass spectrometry.
  • MIC- 1 polypeptides were designed to have increased solubility.
  • solubility was enhanced and stability was improved by modification of the amino acid sequence of the MIC- 1 polypeptide.
  • modification was done within the amino acid sequence of the MIC- 1 polypeptide (in- sequence mutation).
  • MIC- 1 polypeptides with an N-terminal amino acid extension can be expressed in bacteria such as E. coli.
  • large scale protein production of the MIC-1 polypeptides with an N-extension could take of using Inclusion Bodies (IB) as this represent an advantageous approach to controlling process recovery, protein purity, protease degradation and general protein stability. This becomes particular important for large scale protein production.
  • IB Inclusion Bodies
  • N-terminal amino acid_extension F, I, L, M, V, W and Y were excluded, since they could contribute to protein aggregation. H, K, and R were also excluded, since they could cause undesired binding on cell membrane.
  • A, C, E, G, P, S, T, D, N, and Q are preferred for the N-extension sequence. E and D are particularly preferred since they increase the solubility by decreasing pi value of the compound.
  • C could provide a -SH group which can be used for protraction purpose, such as fatty acid conjugation and PEGylation.
  • one or two additional Alanine(s), Glycine(s) or Serine(s) were added at the very N-terminal to increase the initial Methionine removing efficiency when MIC-1 polypeptides with N-extension were expressed in E.coli.
  • N-extensions comprise sequences originating from human proteins (humanized sequences); some comprise artificially designed sequence(s) (e.g. GS, SG, AEE, AES, GEPQ (SEQ ID NO : 123), GEPS (SEQ ID NO : 118)); some comprise several repeats of the humanized sequences or artificial sequences; some comprise a combination of the above.
  • Several 6-residue sequences (6-mers) were designed. N-extensions could comprise one or more of a 6-mers, part of a 6-mers (e.g., 1-5 residues of a 6-mers), or a combination of the above.
  • the amino acid residues of the artificial sequences (including 6-mers) and the humanized sequences could be arranged in any order.
  • 6-mers 6-mer-l SPAGSP (SEQ ID NO :4)
  • Certain internal residues of MIC-1 (SEQ ID NO: l) were modified, e.g. by substitution.
  • a hydrophobic residue of MIC-1 could be substituted with a hydrophilic residue, preferably by with an acidic residue; a positive charged residue could be substituted with an acidic residue, etc.
  • methionine could be substituted with other amino acids, e.g. E, F or L.
  • In-sequence mutations for increasing solubility include but are not limited to : P11E, H 18E, R21E, A30E, A47E, R53E, A54E, M57E, H66E, R67E, L68E, K69E, A75E, A81E, P85E, Q90E, T92E, L105E and K107E.
  • In-sequence mutations for decreasing oxidation include but are not limited to: M43L, M43E, M57E, M57L, M86F and M86L.
  • In-sequence mutations for increasing chemical stability include but are not limited to N3S, N3E, N3A, N3T, N3P, N3G, N3V, N3H, N3Y and N3Q.
  • In-sequence mutations for conjugation include but are not limited to K69R, K107R and K91R.
  • in-sequence mutations include but are not limited to a deletion of N3 (des- N3) and/or a deletion of the first 3 residues. pi calculation
  • the calculated pi of a MIC-1 polypeptide with an N-terminal amino acid extension is defined as the pH at which the net calculated charge of the MIC-1 polypeptide with a N-terminal amino acid extension is zero.
  • the calculated charge of the MIC-1 polypeptide with the N-terminal amino acid extension as a function of pH is obtained using the pKa values of the amino acid residues described in Table 1 and the method described by B. Skoog and A. Wichman (Trends in Analytical Chemistry, 1986, vol. 5, pp. 82-83).
  • the side chain pKa of cysteine (Cys) is only included in the charge calculation for cysteines with a free sulfhydryl group.
  • the N-extension may contain one cysteine mutation.
  • the calculated pi value of human wild type MIC-1 is 8.8 as the homodimer.
  • the calculated pi values of MIC-1 polypeptide are shown in Table 4.
  • Example- 1 Expression and fermentation of the MIC-1 polypeptide or the MIC-1 polypeptide with an N -terminal extension
  • the cDNA of MIC-1 polypeptide or MIC-1 polypeptide with an N-terminal extension was sub-cloned into a pETl lb derived vector.
  • Overexpression of MIC-lf polypeptide or MIC-1 polypeptide with an N-terminal extension as inclusion bodies was induced in E. coli by 0.5mM isopropyl ⁇ -d-thiogalactoside (IPTG) when the cell density reached an OD600 of 1.0.
  • IPTG isopropyl ⁇ -d-thiogalactoside
  • Fermentation was carried out on fed-batch process in chemical defined medium as supplement. Fermentation yield largely depended on different polypeptide, which varied from 1 g/L to 8 g/L from polypeptide to polypeptide.
  • Example-2 Purification and refolding:
  • the MIC-1 polypeptide or MIC-1 polypeptide with an N-terminal extension were further purified as follows:
  • MIC-1 polypeptide or MIC-1 polypeptide with an N-terminal extension was transferred to DPBS, and stored frozen.
  • MIC-1 polypeptides or MIC-1 polypeptides with an N-terminal extension and their maximal solubility at pH8 in Tris buffer are shown in Table 5.
  • Table 5 MIC-1 polypeptides or MIC-1 polypeptides with an N-terminal extension and their maximal solubility at pH8 in Tris buffer tested according to
  • Example-3 pH-dependent solubility of MIC-1 polypeptide with an N-terminal extension
  • MIC-1 polypeptides with N-terminal extensions were dissolved in a mixture of water and ethanol (60% water and 40% ethanol) with a concentration range between 3 mg/ml to 10 mg/ml. The solvent was evaporated with SpeedVac (Concentrator Plus, Eppendorf) for 6 hours to obtain pellet of the MIC-1 polypeptide with N-terminal extension.
  • SpeedVac SpeedVac
  • Buffers were added into each well of the 96-well plate together with the MIC-1 polypeptides with N-terminal extensions.
  • the amount used may not be exactly the same but all targeting a theoretical concentration within 12-18 mg/ml.
  • concentration of MIC-1 polypeptide with N-terminal extension in the supernatant was determined by UPLC (Table 6). Based on the results, solubility of the MIC-1 polypeptide with N-terminal extension of the invention was significantly improved between pH 6-9 compared with wtMIC-1.
  • the optimal pH window of the MIC-1 polypeptides with an N- extension falls into the pH range that is preferred for formulation, e.g. pH 6.5-8.5.
  • the MIC-1 polypeptides with an N- terminal extension were dissolved in a mixture of water and ethanol (60% water and 40% ethanol) with a concentration range between 3 mg/ml to 10 mg/ml. Then the solution (150 ⁇ _ each well) was aliquot into a 96-well plate (Corning). The solvent was evaporated with SpeedVac (Concentrator Plus, Eppendorf) for 6 hours to obtain pellet of the MIC-1 polypeptide with an N-terminal extension. Tris buffer (pH 8.0, without excipients) was added into each well of the 96-well plate. The amount of buffer added to the well was less than the amount needed for solving the whole pellet in the well, so that maximal concentration was achieved.
  • the plate was shaken on a plate shaker at 800 rpm (MixMate, Eppendorf) for 2 hours. The pellet was spun down at 3600 g for 5 min. The supernatants were transferred to a 96-deep-well plate and diluted 20 times with 40% ethanol. Then all of the samples were subject to UPLC (Acquity, Waters), plate reader (Infinite M200 pro, Tecan) and UV spectrometer (NanoDrop 8000, Thermo Scientific) to determine the concentration (Table 7)
  • solubility of the MIC-1 polypeptides with an N-terminal extension of the invention was significantly improved at pH 8.0.
  • the MIC-1 polypeptides with an N-terminal extension achieved solubility of more than 30 mg/ml at pH 8.0.
  • Table 7 Max solubility test of MIC-1 polypeptides with an N-terminal extension at pH 8.0
  • the purpose of this example was to establish a cell based in vitro assay for testing MIC-1 activity.
  • Mammalian cells were transfected and stably expressed full length MIC-1 receptor (hGFRAL) and its full signaling co-receptor hRET51.
  • Plasmids expressing full length hGFRAL and full length hRET51 were constructed by inserting synthesized DNA nucleotides encoding full length hGFRAL and full length hRET51 into mammalian expression vector pEL.
  • IRES internal ribosome entry site
  • IRES internal ribosome entry site
  • Results from screening MIC-1 polypeptides with an N-terminal extension using BHK21-hGFRAL-IRES-hRET is shown in Table 8.
  • Example 7 Effect of MIC-1 polypeptides with an N-terminal extension on food intake in lean Sprague Dawley rats
  • Collected data are expressed as daily food intake (24hour food intake) measured from the onset of each daily 12hour dark phase to the next day dark phase.
  • Daily changes in food intake in response to administrated compound were calculated by subtracting the average daily food intake of the vehicle group from the average daily food intake of the treatment group. Changes were considered significant if p ⁇ 0.1 using a two-tailed student's t-test.
  • Results are expressed as the "maximum reduction” in food intake compared with vehicle (percentage) recorded during the study period.
  • Data are also expressed as the "accumulated reduction" in food intake which as the sum of significant (p ⁇ 0.1) daily reductions in food intake
  • Table 9 Effect of daily doses (8 nmol/kg) of MIC-1 polypeptides with an N- extension on food intake in lean SD rats.
  • SEQ ID NO : 105 and SEQ ID NO : 106 had a maximum and accumulated in vivo efficacy which was 40-50% greater than wtMIC-1 with subcutaneous dosing.
  • the increase in efficacy was furthermore associated with an increase in solubility as compounds according to SEQ ID NO :92, SEQ ID NO : 104, SEQ ID NO : 105 and SEQ ID NO: 106 all had elevated solubility and a significant greater in vivo efficacy compared with wtMIC-1. This correlation seems not to be explained by changes in the in vitro Emax as all compounds in table 8, except compound according to SEQ ID NO : 105, had an Emax comparable with wtMIC-1.
  • compound SEQ ID NO: 105 had a lower Emax than wtMIC-1 and was still more efficacious than wtMIC-1 in vivo. Also, the in vitro potencies were comparable between compounds as none of the compounds had an EC50 which differed from wtMIC-1. Thus, the association between increased in vivo efficacies and increased solubility is surprising and cannot be simply be explained by changes in increased receptor activation in vitro.
  • Example 8 MIC-1 expression and initial Met removal efficiency of different 12- mer blocks
  • N-Formyl-Methionine is recognized by the immune system as foreign material, or as an alarm signal released by damaged cells, and stimulates the body to fight against potential infection (Pathologic Basis of Veterinary Disease5: Pathologic Basis of Veterinary Disease, By James F. Zachary, M. Donald McGavin).
  • Methionine is an instable residue that could be easily oxidized. Therefore, the N- Met cleavage efficiency is very important to MIC-1 expression.
  • the cDNA of MIC-1 polypeptide with N-terminal extension was sub-cloned into a pETl lb derived vector.
  • Overexpression of MIC-1 polypeptides with an N-terminal extension as inclusion bodies or soluble protein was induced in E. coli by 0.5mM isopropyl ⁇ -d-thiogalactoside (IPTG) when the cell density reached an OD600 of 1.0.
  • IPTG isopropyl ⁇ -d-thiogalactoside
  • Fermentation was carried out on fed-batch process in chemical defined medium as supplement. Fermentation yield largely depended on different compounds, which varied from 1 g/L to 8 g/L from compound to compound.
  • N/A means MIC-1 with the N-terminal extension c id not express in E.coli.
  • N/A means MIC-1 po ypeptide with N-terminal extension did not express in E.coli.
  • Example 9 Expression level and inclusion body ratio of MIC-1 polypeptide with 2* or 2.5*12mer N-extension
  • inclusion body is usually considered as a good choice mainly due to its better up-scaling properties, which mainly include: high expression level, simple recovery step and high purity, protease-resistant and good process stability.
  • MIC-1 polypeptides with an N-terminal extension could be expressed either inclusion body or soluble form, which is mainly dependent on compounds' pi and extension length. The results are shown in Table 13 and Fig.4.
  • the solubility of MIC- 1 polypeptides with in-sequence mutations are sh Table 14 and Fig.5 (the MIC- 1 polypeptide sequence is MIC- 1 ⁇ 1-3) .
  • MIC-1 polypeptides initiating with 12mer- (4+2+_), - (4+4+_) and - (4+3+_) were investigated with their ability to express inclusion body. It was shown that the inclusion body ratio is >90% when pl>5.1.
  • MIC-1 polypeptides with in-sequence mutations M57E/H66E mainly expressed soluble fractions.
  • Example 9 Production of MIC-1 polypeptides with an N-terminal extension including a Cys mutation
  • SEQ ID NO: 302 SEPATSGCETPG- S(-23)C 100% 5.7
  • WtMIC-1 homo-dimer contains total of 9 pairs of disulphide bonds and in theory, introducing a new cysteine will disturb the original disulphide bond matching by disulphide bond scrambling, which could further decrease refolding yield. While in our experiments, it is surprising to find that these Cys mutants listed were tested in the same refolding buffer used for wtMIC-1 refolding and showed similar refolding yield ( ⁇ 50% to 60%) as wtMIC-1 or solubility-engineered MIC-1 polypeptide with an N-terminal extension described.
  • Table 16 pH-dependent solubility test of MIC-1 polypeptide with an N-terminal extension including a Cys mutation
  • Table 17 Maximal solubility determination test of MIC-1 polypeptide with an N- terminal extension including a Cys mutation
  • Example 10.1 Preparation of 17-r(SVl-carboxy-3-( 2-f 2- ⁇ 2-f 2- ⁇ 4- formylbenzylcarbamovnmethoxy1ethoxy ethylcarbamovn- methoxy1ethoxy ethylcarbamovnpropylcarbamoyl1heptadecanoic acid
  • the aqueous phase contained the product; therefore it was extracted with
  • dichloromethane (2 x 250 mL) and N,N-dimethylformamide (250 mL). Fmoc group was removed by treatment with 20% piperidine in dimethylformamide (1 x 5 min, 1 x 10 min, 1 x 30 min, 3 x 250 mL). Resin was washed with N,N-dimethylformamide (3 x 250 mL), 2-propanol (2 x 250 mL) and dichloromethane (300 mL, 2 x 250 mL).
  • Resin was filtered and washed with N,N-dimethylformamide (2 x 250 mL), dichloromethane (2 x 250 mL) and N,N-dimethylformamide (250 mL). Fmoc group was removed by treatment with 20% piperidine in dimethylformamide (1 x 5 min, 1 x 10 min, 1 x 30 min, 3 x 250 mL). Resin was washed with N,N-dimethylformamide (3 x 250 mL), 2-propanol (2 x 250 mL) and dichloromethane (300 mL, 2 x 250 mL).
  • Resin was shaken for 1 hour, filtered and washed with N,N-dimethylformamide (3 x 250 mL), dichloromethane (2 x 250 mL), methanol (2 x 250 mL) and dichloromethane (350, 6 x 250 mL).
  • the product was cleaved from resin by treatment with 2,2,2-trifluoethanol (250 mL) for 18 hours.
  • Resin was filtered off and washed with dichloromethane (2 x 250 mL), 2-propanol/dichloromethane mixture (1 : 1, 2 x 250 mL), 2-propanol (250 mL) and dichloromethane (3 x 250 mL).
  • hexafluorophosphate (HATU, 11.4 g, 30.1 mmol) and triethylamine (8.77 mL, 62.9 mmol) were subsequently added to a solution of (S)-22-(tert-butoxycarbonyl)-41,41- dimethyl-10, 19,24,39-tetraoxo-3,6, 12,15,40-pentaoxa-9,18,23-triazadotetracontanoic acid (22.4 g, 27.4 mmol) in dry dichloromethane (110 mL).
  • Triethylamine (5.72 mL, 41.0 mmol) was added to a suspension of (2-amino-ethyl)-carbamic acid benzyl ester hydrochloride (6.94 g, 30.1 mmol) in dry dichloromethane (165 mL) and the resulting mixture was added to the above solution. The mixture was stirred at room temperature overnight, and then it was evaporated to dryness.
  • N,N-Diisopropylethylamine (4.98 mL, 28.6 mmol) was added to a solution of the above amine (6, 20.5 g, 23.8 mmol) in dry dichloromethane (290 mL) at -30 °C under argon.
  • Bromoacetyl bromide (2.48 mL, 28.6 mmol) was added dropwise and the resulting solution was stirred at -30 °C for additional 3 hours. The cooling bath was removed, the mixture was stirred at room temperature for 1 hour, and then the solvent was removed in vacuo.

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JP2020520918A (ja) 2020-07-16
AU2018272911A1 (en) 2019-12-19
PH12019550241A1 (en) 2020-12-07
RU2019141031A3 (es) 2021-07-16
CA3064320A1 (en) 2018-11-29
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CN111183148A (zh) 2020-05-19
US20180339057A1 (en) 2018-11-29
US20200079829A1 (en) 2020-03-12
KR20200010353A (ko) 2020-01-30
US10398782B2 (en) 2019-09-03
BR112019024102A2 (pt) 2020-06-02
WO2018215525A1 (en) 2018-11-29
JP7015322B2 (ja) 2022-02-02
MX2019013801A (es) 2020-01-30
AU2018272911B2 (en) 2022-09-08
TW201900219A (zh) 2019-01-01
PE20200514A1 (es) 2020-03-05
MA48956A (fr) 2020-04-08
CL2019003441A1 (es) 2020-05-08

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